Silver nanoparticles induce premutagenic DNA oxidation that can be prevented by phytochemicals from Gentiana asclepiadea.

Among nanomaterials, silver nanoparticles (AgNPs) have the broadest and most commercial applications due to their antibacterial properties, highlighting the need for exploring their potential toxicity and underlying mechanisms of action. Our main aim was to investigate whether AgNPs exert toxicity by inducing oxidative damage to DNA in human kidney HEK 293 cells. In addition, we tested whether this damage could be counteracted by plant extracts containing phytochemicals such as swertiamarin, mangiferin and homoorientin with high antioxidant abilities. We show that AgNPs (20 nm) are taken up by cells and localised in vacuoles and cytoplasm. Exposure to 1, 25 or 100 µg/ml AgNPs leads to a significant dose-dependent increase in oxidised DNA base lesions (8-oxo-7,8-dihydroguanine or 8-oxoG) detected by the comet assay after incubation of nucleoids with 8-oxoG DNA glycosylase. Oxidised DNA base lesions and strand breaks caused by AgNPs were diminished by aqueous and methanolic extracts from both haulm and flower of Gentiana asclepiadea.

Gentiana asclepiadea exerts antioxidant activity and enhances DNA repair of hydrogen peroxide- and silver nanoparticles-induced DNA damage.

Exposure to high levels of different environmental pollutants is known to be associated with induction of DNA damage in humans. Thus DNA repair is of great importance in preventing mutations and contributes crucially to the prevention of cancer. In our study we have focused on quantitative analysis of Gentiana asclepiadea aqueous or methanolic extracts obtained from flower and haulm, their antioxidant potency in ABTS post-column derivatisation, and their potential ability to enhance DNA repair in human lymphocytes after hydrogen peroxide (H(2)O(2)) treatment (250 µM, 5 min). We also studied DNA repair in human kidney HEK 293 cells after exposure to 20 nm silver nanoparticles (AgNPs) (100 µg/ml, 30 min) in the presence and absence of the plant extract. We have found that mangiferin along with unidentified polar compounds are the most pronounced antioxidants in the studied extracts. Extract from haulm exhibited slightly stronger antioxidant properties compared to flower extracts. However, all four extracts showed significant ability to enhance DNA repair in both cell types after H(2)O(2) and AgNP treatments.

Impact of agglomeration and different dispersions of titanium dioxide nanoparticles on the human related in vitro cytotoxicity and genotoxicity.

The published results on nanoparticles cytotoxicity and genotoxicity such as titanium dioxide nanoparticles (TiO(2) NPs) are inconsistent, and often conflicting and insufficient. Since different parameters may have impact on the toxicity results, there is need to lay stress on detailed characterization of NPs and the use of different testing conditions for assessment of NPs toxicity. In order to investigate whether dispersion procedures influence NP cytotoxicity and genotoxicity, we compared two protocols giving TiO(2) NP dispersions with different stability and agglomeration states. Detailed primary and secondary characteristics of both TiO(2) NP dispersions in culture media were carried out before toxicological testing; TK6 human lymphoblast cells, EUE human embryonic epithelial cells and Cos-1 monkey kidney fibroblasts were used to assess cytotoxicity (by trypan blue exclusion, proliferation activity and plating efficiency assays) and genotoxicity (by the comet assay). DNA strand breaks were detected by the alkaline comet assay. DNA oxidation lesions (especially 8-oxo-7,8-dihydroguanine, 8-oxoG) were measured with a modified comet assay including incubation with specific repair enzyme formamidopyrimidine DNA glycosylase (FPG). The TiO(2) NPs dispersion with large agglomerates (3 min sonication and no serum in stock solution) induced DNA damage in all three cell lines, while the TiO(2) NPs dispersed with agglomerates less than 200 nm (foetal serum in stock solution and sonication 15 min) had no effect on genotoxicity. An increased level of DNA oxidation lesions detected in Cos-1 and TK6 cells indicates that the leading mechanism by which TiO(2) NPs trigger genotoxicity is most likely oxidative stress. Our results show that the dispersion method used can influence the results of toxicity studies. Therefore at least two different dispersion procedures should be incorporated into assessment of cyto- and genotoxic effects of NPs. It is important, when assessing the hazard associated with NPs, to establish standard testing procedures and thorough strategies to consider the diverse conditions relevant to possible exposures.

Gentiana asclepiadea protects human cells against oxidation DNA lesions.

The objectives of this study were to examine whether the methanolic and aqueous extracts from the haulm and flower of Gentiana asclepiadea exhibited free radical scavenging and protective (antigenotoxic) effect against DNA oxidation induced by H(2)O(2) in human lymphocytes and human embryonic kidney cells (HEK 293). All four extracts exhibited high scavenging effect on 1,1-diphenyl-2-picrylhydrazyl radicals at concentrations 2.5 and 25 mg ml(-1). The level of DNA damage was measured using the alkaline version of single-cell gel electrophoresis (comet assay). Challenge with H(2)O(2) shows that the pre-treatment of the cells with non-genotoxic doses of Gentiana extracts protected human DNA-either eliminated or significantly reduced H(2)O(2) induced DNA damage. The genotoxic activity of H(2)O(2) was most effectively decreased after 30 min of pre-incubation with 0.05 mg ml(-1) (range, 93.5%-96.3% of reduction in lymphocytes) and 0.25 mg ml(-1) (range, 59.5%-71.4% and 52.7%-66.4% of reduction in lymphocytes and HEK 293 cells, respectively) of G. asclepiadea extracts. These results suggest that the tested G. asclepiadea extracts could be considered as an effective natural antioxidant source.

DNA alkylation lesions and their repair in human cells: modification of the comet assay with 3-methyladenine DNA glycosylase (AlkD).

3-methyladenine DNA glycosylase (AlkD) belongs to a new family of DNA glycosylases; it initiates repair of cytotoxic and promutagenic alkylated bases (its main substrates being 3-methyladenine and 7-methylguanine). The modification of the comet assay (single cell gel electrophoresis) using AlkD enzyme thus allows assessment of specific DNA alkylation lesions. The resulting baseless sugars are alkali-labile, and under the conditions of the alkaline comet assay they appear as DNA strand breaks. The alkylating agent methyl methanesulfonate (MMS) was used to induce alkylation lesions and to optimize conditions for the modified comet assay method with AlkD on human lymphoblastoid (TK6) cells. We also studied cellular and in vitro DNA repair of alkylated bases in DNA in TK6 cells after treatment with MMS. Results from cellular repair indicate that 50% of DNA alkylation is repaired in the first 60 min. The in vitro repair assay shows that while AlkD recognises most alkylation lesions after 60 min, a cell extract from TK6 cells recognises most of the MMS-induced DNA adducts already in the first 15 min of incubation, with maximum detection of lesions after 60 min' incubation. Additionally, we tested the in vitro repair capacity of human lymphocyte extracts from 5 individuals and found them to be able to incise DNA alkylations in the same range as AlkD. The modification of the comet assay with AlkD can be useful for in vitro and in vivo genotoxicity studies to detect alkylation damage and repair and also for human biomonitoring and molecular epidemiology studies.

Cytotoxic and genotoxic effect of methanolic flower extract from Gentiana asclepiadea on COS 1 cells.

OBJECTIVE: The purpose of this study was to assess whether a methanol extract isolated from the flower of Gentiana asclepiadea had potential cytotoxic or genotoxic effect on COS 1 (monkey kidney) cell line. Five various concentrations of the extract were investigated for cytotoxicity and genotoxicity and to determine non-cytotoxic and non-genotoxic concentrations suitable for utilization in pharmacology and medicine. METHODS: Cytotoxicity was determined using the proliferation (growth activity) and the plating efficiency (colony forming ability) assays after 24 hour incubation of COS 1 cells with different concentrations of methanolic flower extract from G. asclepiadea. To assess potential genotoxicity, the comet assay or SCGE (Single-Cell Gel Electrophoresis) was used. RESULTS: We found that only the highest (5 and 25 mg/ml) concentrations of the extract revealed cytotoxic and genotoxic effect. We have also determined concentrations that stimulated cell growth (0.25 mg/ml) and colony forming ability (0.25-2.5 mg/ml) and did not exhibit genotoxic effect (0.25-2.5 mg/ml). CONCLUSIONS: We found out that extract of G. asclepiadea was neither cytotoxic nor genotoxic in a wide range of concentrations (0.25-2.5 mg/ml) and thus can be used to further investigate potential beneficial usage in pharmacology and medicine.

Potential antioxidant activity, cytotoxic and apoptosis-inducing effects of Chelidonium majus L. extract on leukemia cells.

OBJECTIVES: The purpose of this study was to assess whether a methanol extract isolated from the greater celandine Chelidonium majus L. (CME) had antioxidant effect and was able to inhibit proliferation and to induce apoptosis in leukemia cells in vitro. METHODS: The potential antioxidant activity of CME was proved by the 1,1-diphenyl- 2-picrylhydrazyl (DPPH) radical scavenging assay. The cytotoxicity of CME was measured by the cell growth inhibition assay using murine leukemia L1210 cell line and human promyelocytic HL-60 leukemia cells. Apoptosis-inducing effect was determined by fluorescence microscopy (chromatin condensation and nuclear DNA fragmentation). RESULTS: In the DPPH assay CME acted as a scavenger of DPPH free radical. The results on antiproliferative properties assessment clearly demonstrated that CME had a cytotoxic effect towards both leukemia cell lines in a dose-dependent manner. In addition, the human promyelocytic HL-60 cells were more sensitive to CME treatment than the L1210 cells. CONCLUSIONS: We concluded that the extract of C. majus L. had a strong antioxidant potential and exerted the antiproliferative activity via apoptosis on leukemia cells. CME due to the presence of the isoquinoline alkaloids and the flavonoid components may play an important role in both cancer chemoprevention through its antioxidant activity and modern cancer chemotherapy as cytotoxic and apoptosis-inducing agent.